Disease relevance of rare VPS13B missense variants for neurodevelopmental Cohen syndrome

Autosomal recessive Cohen syndrome is a neurodevelopmental disorder characterized by postnatal microcephaly, intellectual disability, and a typical facial gestalt. Genetic variants in VPS13B have been found to cause Cohen syndrome, but have also been linked to autism, retinal disease, primary immunodeficiency, and short stature. While it is well established that loss-of-function mutations of VPS13B cause Cohen syndrome, the relevance of missense variants for the pathomechanism remains unexplained. Here, we investigate their pathogenic effect through a systematic re-evaluation of clinical patient information, comprehensive in silico predictions, and in vitro testing of previously published missense variants. In vitro analysis of 10 subcloned VPS13B missense variants resulted in full-length proteins after transient overexpression. 6/10 VPS13B missense variants show reduced accumulation at the Golgi complex in the steady state. The overexpression of these 6/10 VPS13B missense variants did not rescue the Golgi fragmentation after the RNAi-mediated depletion of endogenous VPS13B. These results thus validate 6/10 missense variants as likely pathogenic according to the classification of the American College of Medical Genetics through the integration of clinical, genetic, in silico, and experimental data. In summary, we state that exact variant classification should be the first step towards elucidating the pathomechanisms of genetically inherited neuronal diseases.


Summary on literature reported disease-associated VPS13B missense variants
Six out of 29 disease-associated VPS13B missense variants were reported in compound heterozygosity or homozygosity with two further most likely truncating variants on both alleles (Lys1129Arg, Asp1210Tyr, Thr1289Ser, Ile2795Thr, Ser3303Arg, and Val3420Met. Thus, those rare missense variants can be suspected to be more likely benign [33,34]. Five out of 29 disease-associated VPS13B missense variants were reported in compound heterozygosity with one most likely truncating variant on the second allele (Trp185Arg, Thr1068Ile, Leu2168, Tyr2316Cys, and Gly2704Arg).
Seven out of 29 disease-associated VPS13B missense variants were reported in different cases as homozygous variants (Ser824Ala, Ile1611Asn, Val2456Ile, Gly2620Asp, Ser2748, Asn2968Ser, and Arg3198Trp). Asn2968Ser was originally identified in a consanguine family in homozygosity [20]. However, the same mutation was more recently in compound heterozygosity with another missense variant Leu2821Ile [29]. For the missense variant Arg3198Trp in autism conflicting genetic data have been reported [15]. This variant is inherited by both male children in homozygosity from an unaffected homozygous father and an unaffected heterozygous mother in one family, and by another male patient in heterozygosity from an unaffected heterozygous father and an unaffected heterozygous mother in another family. For the latter family no further VPS13B-associated variation was identified.
Six out of 29 disease-associated VPS13B missense variants were reported in heterozygous inheritance where a second variant is still unknown (Phe274Val, Ala590Thr, Thr1289Ala, Lys1682Glu, Asn3088Tyr, and Pro3962Arg). Here, the three missense variants Phe274Val, Thr1289Ala, and Leu1682Glu) have been reported in targeted NGS for autism or primary immunodeficiency disease screens in occurrence with other heterozygous variants in further disease-causing genes [4,21].The results from in vitro characterization of the hitherto cloned VPS13B missense variations and their update on ACMG classification are summarized in table 1.

Details on literature reported disease-associated VPS13B missense variants
The missense variant Trp185Arg was identified in one male with intellectual disability, microcephaly and joint hypermobility; combining typical features of Cohen syndrome [36]. At the age of 3 years clinical assessment did not reveal any ophthalmic or blood problems. The missense variant was detected as compound heterozygous with the fatal missense variant Cys733*.
The missense variant Phe274Val was identified in a male patient and was found in heterozygosity, lacking further variants in VPS13B [21]. The patient belonged to a study in which target gene enrichment of autism-associated genes occurred. However, a second heterozygous missense variant was identified in RAI1. The authors state that this missense variation is most unlikely disease causing.
The missense variant Gly567Glu was identified in compound heterozygosity with another Pro1133Ser missense variant during diagnostic family-based exome sequencing [11]. All patients were found to have a genetic etiology but lack a genetic diagnosis. Functional follow-up evidence of the hitherto identified missense variants remains elusive.
The missense variant Ala590Thr was identified as heterozygous in one female offspring from nonconsanguineous Italian parents [18]. A compound heterozygous second variant in VPS13B is missing so far. Age of clinical assessment was 24 years. The Cohen syndrome phenotype is incomplete without documented microcephaly, retinopathy and myopia. However, moderate intellectual disability and neutropenia indicate a Cohen syndrome.
The missense variant Ser824Ala was identified in a male patient with incomplete Cohen syndrome phenotype [37]. The analysis was performed in a screening of genetic causes for autism and was found in homozygous state. The authors speculate an incomplete, hypomorph Cohen syndrome due to reduced protein availability.
The missense variant Thr1068Ile was identified in compound heterozygosity with a mild intragenic c.8016+7G > C, which has not been tested for its effect on the splicing event [36]. The major phenotype of the patient were repeated seizures and very mild intellectual disability.
The missense variant Lys1129Arg was identified in two siblings with Cohen syndrome [33]. However, both affected children carrier two compound heterozygous LoF variations in VPS13B, which segregate within the family and were therefore considered as solely responsible to cause Cohen syndrome. In a more recent study this missense variant reappeared in a genetic approach on patients with retinal disease [35]. The respective patient has a compound heterozygous pathogenic variant in VPS13B (c.10232delC) and was clinically assessed to have Cohen syndrome.
The missense variant Pro1133Ser was identified in compound heterozygosity with the above described Gly567Glu missense variant [11].
The heterozygous missense variant Asp1210Tyr was identified in a whole-genome sequencing approach on patients with retinal disease [3]. The respective patient presents with two other compound heterozygous pathogenic variants in VPS13B (Ser864* and Met2124Valfs*44), probably solely causative for the inherited retinal disease.
The heterozygous missense variant Thr1289Ala occurred in a targeted disease genes approach for primary immunodeficiency [4]. A second variant in VPS13B could not be identified; however, further heterozygous variants were identified in the respective patient in other primary immunodeficiencyassociated disease genes: SLC37A4, SCNN1G, and CXCR4.
The missense variant Thr1289Ser in the second child of a nonconsanguineous Caucasian African couple was identified in combination with a paternal inherited 3 bp insertion (c. 11752_11753insATG) and a maternal inherited 315 kb deletion spanning from exon 4 of the OSR2 gene to exon 17 of the VPS13B gene (chr8: 100015029…100347846del) [30]. Phenotypic assessment occurred at the age of 2 years with Cohen syndrome-like facial dysmorphism but without typical ophthalmological and/or hematological Cohen syndrome-associated findings.
The homozygous missense variant Ile16111Asn was recognized in Moroccan twins from consanguineous parents [9]. Both twins show typical Cohen syndrome-like features including intellectual disability, microcephaly, facial dysmorphism, and slender extremities.
The heterozygous missense variant Lys1682Glu occurred in a targeted disease genes approach for primary immunodeficiency [4]. A second variant in VPS13B could not be identified; however, further heterozygous variants were identified in the respective patient in other primary immunodeficiencyassociated disease genes: ADA, FERMT3, and CD79B.
The compound heterozygous missense variant Leu2168Arg was found in combination with a 2 bp deletion (c.3348_3349delCT) in a finish patient with typical a finish phenotype of Cohen syndrome [19].
The missense variant Tyr2316Cys was identified as compound-heterozygous together with a nonsense variation (c.11240C>T) [13]. Clinical assessment of the 4-yearold female showed typical facial dysmorphism, intellectual disability and postnatal microcephaly. However, ophthalmological and hematological findings were unremarkable which is most likely due to the younger age of the patient at clinical assessment.
The homozygous missense variant Val2456Ile was identified in an exome sequencing approach in persons with severe intellectual disability [7]. The male patient was clinically assessed at the age of 4 years and presents with intellectual disability and microcephaly but normal motor development. MRI of the brain was normal, and at the age of around one year he lost his previously accomplished communication skills.
The missense variant Gly2620Asp was identified in two consanguine families from the Oman. All patients share a typical facial dysmorphism, intellectual disability, myopia and microcephaly in agreement with Cohen syndrome [13,26]. Moreover, the oldest child (10 years at clinical assessment) showed initial ophthalmological abnormalities indicative of retinopathy and her MRT screening showed a comparable enlarged corpus callosum [26].
The missense variant Gly2704Arg was identified in a screening of genetic causes for autism [37]. However, the clinical reevaluation of the patient showed typical Cohen syndrome-like features. Moreover, the missense variant was identified to be compound heterozygous with a nonsense variation in VPS13B (c.8110G>A).
The missense variant Ser2748Leu was identified in two consanguineous families [34]. All three patients (two female, one male) showed typical Cohen syndrome-like facial appearance and intellectual disability. Two patients presented also with postnatal microcephaly. Due to their younger age (8, 5 and 2.5 years) the exclusion of later onset of ophthalmological problems could not be excluded.
The missense variant Ile2795Thr was recognized in 8 patients from an Amish community in US, Ohio [10]. However, all 8 patients carry an additional homozygous insertion (c.9183_9184insT) in VPS13B, which is most likely solely causative for the typical Cohen syndrome phenotype in all those patients. In another clinical study on bilateral angle closure glaucoma the missense variant Ile2795Thr was identified in heterozygosity in the described patient with Cohen syndrome. The patient has Amish background and was further genetically tested carrying the homozygous c.9258_9259insT [24].
The missense variant Leu2821Ile was detected in a whole-exome sequencing screen across clinical indicators with focus on family-based analysis and classified as very likely pathogenic [29]. However, from the supported material, one cannot clearly extract status of heterozygosity, inheritance or clinical presentation of the belonging patient.
The missense variant Asn2968Ser was identified in homozygous occurrence in Belgian twins [20]. Both patients presented with a typical Cohen syndrome including intellectual disability, microcephaly, neutropenia, retinopathy and myopia. However, the authors themselves speculated that this missense variant could represent a rare nonpathogenic change. This missense variant reoccurred in in a wholeexome sequencing screen across clinical indicators with focus on family-based analysis [29]. However, from the supported material, one cannot clearly extract status of heterozygosity, inheritance or clinical presentation of the belonging patient. In a large-scale genomic WES project on 267 genomes representing the healthy Spanish population the missense variant Asn2968Ser occurred one time on one allele [8].
The heterozygous missense variant Asn3088Tyr was identified in a genetic research study on short stature by target/whole exome sequencing [14]. A second variant in VPS13B was not identified. In addition to the short stature, the female patient was born with an atrial septal defect and mitral regurgitation and presents with intellectual disability as well as microcephaly. However, according to the ACMG guidelines, the authors categorized this missense variant as variance of uncertain significance.
The missense variant Arg3198Trp was identified in a sequenced-based study on identification of rare causal variants in Cohen syndrome and autism [15]. The variant appeared in 2 families, each with 2 affected siblings. The total allele count was 5 in affected individuals versus one in unaffected individuals. Reevaluation occurred by Sanger sequencing. However, the transmission showed also homozygosity in the unaffected father of one family.
The missense variant Val3445Met (c.10333G>A) was identified in heterozygosity with a homozygous Tyr413* (c.1239T>G) by whole-exome sequencing and was confirmed by Sanger sequencing. [23]. The patient presented with Cohen syndrome-agreeable features including normal head circumference at birth and postnatal microcephaly, intellectual disability and a typical facial appearance.
The missense variant Thr3602Ile (c.10805C>T) was identified on same allele in very close proximity to a deletion c.10808_10825delCGAGGCAGCTTGTGCACG [9]. Thus, it is more likely that both alterations occurred at the same DNA changing event. However, they were found to be compound heterozygous with nonsense variation (Arg692*). All patients with VPS13B variations had intellectual disability, a Cohen syndrome-typical facial dysmorphism, microcephaly and slender extremities with narrow hand/feet. Moreover, 11/12 patients had neutropenia.
The missense variants Ser3303Arg and Ala3691Thr were identified as compound heterozygous in one patient with an autisms-like phenotype [37]. However, reevaluation showed Cohen-syndrome-like facial dysmorphism but normocephalic brain development.
The heterozygous missense variant Pro3962Arg was identified in a male patient, lacking a second VPS13B-associated variant [21]. The patient belonged to a study in which target gene enrichment of autism-associated genes occurred. The authors state that this missense variation is most unlikely disease causing.
Genotype and phenotypical characteristics of these previously reported patients are summarized in supplementary table T1.